Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

Sensitization of enteric neurons to morphine by HIV-1 Tat protein.

BACKGROUND: Gastrointestinal (GI) dysfunction is a major cause of morbidity in acquired immunodeficiency syndrome (AIDS). HIV-1-induced neuropathogenesis is significantly enhanced by opiate abuse, which increases proinflammatory chemokine/cytokine release, the production of reactive species, glial reactivity, and neuronal injury in the central nervous system. Despite marked interactions in the gut, little is known about the effects of HIV-1 in combination with opiate use on the enteric nervous system. METHODS: To explore HIV-opiate interactions in myenteric neurons, the effects of Tat ± morphine (0.03, 0.3, and 3 µM) were examined in isolated neurons from doxycycline- (DOX-) inducible HIV-1 Tat(1-86) transgenic mice or following in vitro Tat 100 nM exposure (>6 h). KEY RESULTS: Current clamp recordings demonstrated increased neuronal excitability in neurons of inducible Tat(+) mice (Tat+/DOX) compared to control Tat-/DOX mice. In neurons from Tat+/DOX, but not from Tat-/DOX mice, 0.03 µM morphine significantly reduced neuronal excitability, fast transient and late long-lasting sodium currents. There was a significant leftward shift in V(0.5) of inactivation following exposure to 0.03 µM morphine, with a 50% decrease in availability of sodium channels at -100 mV. Similar effects were noted with in vitro Tat exposure in the presence of 0.3 µM morphine. Additionally, GI motility was significantly more sensitive to morphine in Tat(+) mice than Tat(-) mice. CONCLUSIONS & INFERENCES: Overall, these data suggest that the sensitivity of enteric neurons to morphine is enhanced in the presence of Tat. Opiates and HIV-1 may uniquely interact to exacerbate the deleterious effects of HIV-1-infection and opiate exposure on GI function.

Caspase-3 activity is reduced after spinal cord injury in mice lacking dynorphin: differential effects on glia and neurons.

Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin(-/-) mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin(-/-) mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3-4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6-7 mm, there were fewer caspase-3(+) oligodendrocytes and astrocytes than at 3-4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin(-/-) oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg(6)-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.

Glial-restricted precursors: patterns of expression of opioid receptors and relationship to human immunodeficiency virus-1 Tat and morphine susceptibility in vitro.

Recent evidence suggests that human immunodeficiency virus (HIV)-induced pathogenesis is exacerbated by opioid abuse and that the synergistic toxicity may result from direct actions of opioids in immature glia or glial precursors. To assess whether opioids and HIV proteins are directly toxic to glial-restricted precursors (GRPs), we isolated neural stem cells from the incipient spinal cord of embryonic day 10.5 ICR mice. GRPs were characterized immunocytochemically and by reverse transcriptase-polymerase chain reaction (RT-PCR). At 1 day in vitro (DIV), GRPs failed to express mu opioid receptors (MOR or MOP) or kappa-opioid receptors (KOR or KOP); however, at 5 DIV, most GRPs expressed MOR and KOR. The effects of morphine (500 nM) and/or Tat (100 nM) on GRP viability were assessed in GRPs at 5 DIV by examining the apoptotic effector caspase-3 and cell viability (ethidium monoazide exclusion) at 96 h following continuous exposure. Tat or morphine alone or in combination caused significant increases in GRP cell death at 96 h, but not at 24 h, following exposure. Although morphine or Tat caused increases in caspase-3 activity at 4 h, this was not accompanied with increased cleaved caspase-3 immunoreactive or ethidium monoazide-positive dying cells at 24 h. The results indicate that prolonged morphine or Tat exposure is intrinsically toxic to isolated GRPs and/or their progeny in vitro. Moreover, MOR and KOR are widely expressed by Sox2 and/or Nkx2.2-positive GRPs in vitro and the pattern of receptor expression appears to be developmentally regulated. The temporal requirement for prolonged morphine and HIV-1 Tat exposure to evoke toxicity in glia may coincide with the attainment of a particular stage of maturation and/or the development of particular apoptotic effector pathways and may be unique to spinal cord GRPs. Should similar patterns occur in vivo then we predict that immature astroglia and oligodendroglia may be preferentially vulnerable to HIV-1 infection or chronic opiate exposure.

Molecular targets of opiate drug abuse in neuroAIDS.

Opiate drug abuse, through selective actions at mu-opioid receptors (MOR), exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the CNS by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Neurons are affected directly and indirectly by opiate-HIV interactions. Although most opiates drugs have some affinity for kappa (KOR) and/or delta (DOR) opioid receptors, their neurotoxic effects are largely mediated through MOR. Besides direct actions on the neurons themselves, opiates directly affect MOR-expressing astrocytes and microglia. Because of their broad-reaching actions in glia, opiate abuse causes widespread metabolic derangement, inflammation, and the disruption of neuron-glial relationships, which likely contribute to neuronal dysfunction, death, and HIV encephalitis. In addition to direct actions on neural cells, opioids modulate inflammation and disrupt normal intercellular interactions among immunocytes (macrophages and lymphocytes), which on balance further promote neuronal dysfunction and death. The neural pathways involved in opiate enhancement of HIV-induced inflammation and cell death, appear to involve MOR activation with downstream effects through PI3-kinase/Akt and/or MAPK signaling, which suggests possible targets for therapeutic intervention in neuroAIDS.

Differential involvement of p38 and JNK MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite degeneration in striatal neurons.

The role of p38 and c-jun-N-terminal kinases 1/2, members of the mitogen-activated protein kinase family, in mediating the toxic effects of human immunodeficiency virus-1 transactivator of transcription (Tat) and gp120 were explored in primary mouse striatal neurons in vitro. Both Tat and gp120 caused significant increases in p38 and c-jun-N-terminal kinase mitogen-activated protein kinase phosphorylation, caspase-3 activity, neurite losses and cell death in striatal neurons. Tat-induced increases in caspase-3 activity were significantly attenuated by an inhibitor of c-jun-N-terminal kinase (anthra[1,9-cd]pyrazol-6(2H)-one), but not by an inhibitor of p38 ([4-(4-fluorophenyl)-2-(4-methylsul-finylphenyl)-5-(4-pyridyl)1 H-imidazole]), mitogen-activated protein kinase. However, despite preventing increases in caspase-3 activity, c-jun-N-terminal kinase inhibition failed to avert Tat-induced neuronal losses suggesting that the reductions in caspase-3 activity were insufficient to prevent cell death caused by Tat. Alternatively, gp120-induced increases in caspase-3 activity, neurite losses and neuronal death were prevented by p38, but not c-jun-N-terminal kinase, mitogen-activated protein kinase inhibition. Our findings suggest that gp120 induces neuronal dysfunction and death through actions at p38 mitogen-activated protein kinase, while Tat kills neurons through actions that are independent of p38 or c-jun-N-terminal kinase mitogen-activated protein kinase, or through the concurrent activation of multiple proapoptotic pathways.

Genetic background regulates semaphorin gene expression and epileptogenesis in mouse brain after kainic acid status epilepticus.

The host response to neural injury, which can include axonal sprouting and synaptic reorganization is likely to be under tight genetic regulatory control at the level of the genome and may be implicated in epileptogenesis. Despite its importance, however, the molecular basis of synaptic reorganization is unclear. We have studied the development of synaptic reorganization, semaphorin gene expression, and epileptogenesis in hippocampus of epileptogenic sensitive (FVB/NJ) and epileptogenic resistant (C57BL/6J) mice (i.e. distinct genetic backgrounds) after kainic acid-induced status epilepticus. Our results support the hypothesis that disruption of transcriptional regulation of axon guidance genes leads to a differential loss of tonic neuropilin-2 dependent activation of semaphorin 3F receptors on hippocampal neurons on distinct genetic backgrounds. This results in rearranged synaptic circuitry and thus promotes epileptogenesis. These findings may define biologic principles underlying the role of semaphorin signaling which may broadly apply to other systems undergoing neural regeneration.

Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation.

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

Secretoneurin promotes pertussis toxin-sensitive neurite outgrowth in cerebellar granule cells.

The neuropeptide secretoneurin (SN) is an endoproteolytic product of the chromogranin secretogranin II. We investigated the effects of SN on the differentiation of immature cerebellar granule cells derived from the external granular layer (EGL). Secretoneurin caused concentration-dependent increases in neurite outgrowth, reflecting differentiation. The maximum effect was reached at a concentration of 100 nm SN. Secretoneurin immunoneutralization using specific antiserum significantly decreased neurite outgrowth; however, neurite morphology was altered. An affinity chromatography-purified antibody significantly inhibited the outgrowth response to SN (p < 0.001) without altering the morphology. Binding studies suggest the existence of specific G-protein-coupled receptors on the surface of monocytes that recognize SN. Assuming that SN promotes neurite outgrowth in EGL cells by acting through a similar G-protein-coupled mechanism, we treated SN-stimulated EGL cultures with pertussis toxin. Exposure to pertussis toxin (0.1 micro g/mL) showed a significant inhibition of the SN-induced outgrowth. To establish a second messenger pathway we used the protein kinase C inhibitor staurosporine. We found that EGL cell viability was not enhanced following chronic SN treatment for 24 h. These data indicate that SN is a novel trophic substance that can affect cerebellar maturation, primarily by accelerating granule cell differentiation through a signalling mechanism that is coupled to pertussis toxin-sensitive G-proteins.

Dynorphin A toxicity in striatal neurons via an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor mechanism.

Dynorphin A (1-17) is an endogenous opioid peptide that is antinociceptive at physiological concentrations, but in excess can elicit a number of pathological effects. Both kappa-opioid and N-methyl-D-aspartate receptor antagonists modulate dynorphin toxicity, suggesting that dynorphin is acting directly or indirectly through these receptor types. We found in spinal cord neurons that the neurotoxic effects of dynorphin A and several dynorphin-derived peptide fragments are largely mediated by N-methyl-D-aspartate receptors. Despite these findings, aspects of dynorphin A toxicity could not be accounted for by opioid or N-methyl-D-aspartate receptor mechanisms. To address this issue, neurons enriched in kappa-opioid, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors were isolated from embryonic day-15 mouse striata and the effects of extracellularly administered dynorphin A (1-17) and (13-17) on neuronal survival were examined in vitro. Unlike spinal cord neurons, N-methyl-D-aspartate receptors mature later than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors in striatal neurons, thus providing a strategy to elucidate non-N-methyl-D-aspartate receptor-mediated mechanisms of toxicity. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A (1-17 or 13-17; 10 microM) caused significant neuronal losses after 48 to 72 hours versus untreated controls. Dynorphin A or A (13-17) toxicity was unaffected by the opioid receptor antagonist naloxone (10 microM) or by dizocilpine (10 microM). In contrast, the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline- 2,3-dione (10 microM) significantly attenuated only dynorphin A (1-17)-induced neuronal losses and not that induced by dynorphin A (13-17). Dynorphin A (1-17) toxicity was accompanied by a proportional loss of R2 and R3 subunits of the AMPA receptor complex, but not non-N-methyl-D-aspartateR1, expressing neurons and was mimicked by the ampakine 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine. Although it is unclear whether dynorphin A activates alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors directly or indirectly via glutamate release, our culture conditions do not support glutamate retention or accumulation. Our findings suggest that dynorphin A (1-17) can exert toxic effects on striatal neurons via an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor mechanism.

Opioid system diversity in developing neurons, astroglia, and oligodendroglia in the subventricular zone and striatum: impact on gliogenesis in vivo.

Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2-5-day-old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express mu-, delta-, and/or kappa-receptor immunoreactivity in a cell type-specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5-bromo-2'-deoxyuridine (BrdU) incorporation into glial and nonglial precursors. Morphine (a preferential mu-agonist) significantly decreased the number of BrdU-labeled GFAP(+) cells compared with controls or mice co-treated with naltrexone plus morphine. Alternatively, in S100beta(+) cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between mice treated with morphine and those treated with morphine plus naltrexone. Most cells were GFAP(-)/S100beta(-). When BrdU incorporation was assessed within the total population (glia and nonglia), morphine had no net effect, but naltrexone alone markedly increased BrdU incorporation. This finding suggests that DNA synthesis in GFAP(-)/S100beta(-) cells is tonically suppressed by endogenous opioids. Assuming that S100beta and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors.

Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms.

Dynorphin A, a prodynorphin-derived peptide, is able to induce neurological dysfunction and neuronal death. To study dynorphin cytotoxicity in vitro, prodynorphin-derived peptides were added into the culture medium of nonneuronal and neuronal cells or delivered into these cells by lipofection or electroporation. Cells were unaffected by extracellular exposure when peptides were added to the medium. In contrast, the number of viable cells was significantly reduced when dynorphin A or "big dynorphin," consisting of dynorphins A and B, was transfected into cells. Big dynorphin was more potent than dynorphin A, whereas dynorphin B; dynorphin B-29; [Arg(11,13)]-dynorphin A(-13)-Gly-NH-(CH(2))(5)-NH(2), a selective kappa-opioid receptor agonist; and poly-l-lysine, a basic peptide more positively charged than big dynorphin, failed to affect cell viability. The opioid antagonist naloxone did not prevent big dynorphin cytotoxicity. Thus, the toxic effects were structure selective but not mediated through opioid receptors. When big dynorphin was delivered into cells by lipofection, it became localized predominantly in the cytoplasm and not in the nuclei. Big dynorphin appeared to induce toxicity through an apoptotic mechanism that may involve synergistic interactions with the p53 tumor-suppressor protein. It is proposed that big dynorphin induces cell death by virtue of its net positive charge and clusters of basic amino acids that mimic (and thereby perhaps interfere with) basic domains involved in protein-protein interactions. These effects may be relevant for a pathophysiological role of dynorphins in the brain and spinal cord and for control of death of tumor cells, which express prodynorphin at high levels.

Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development.

Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.

Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine.

BACKGROUND: Human immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness. To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat. RESULTS: Acute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors. CONCLUSION: Our observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

Chronic nicotine exposure reduces N-methyl-D-aspartate receptor-mediated damage in the hippocampus without altering calcium accumulation or extrusion: evidence of calbindin-D28K overexpression.

Neuronal accumulation of excess Ca2+ has been implicated in cellular death following several forms of physical and chemotoxic insult. Recent studies have suggested that exposure to agonists at brain nicotinic acetylcholine receptors reduces cytotoxic consequences of increased intracellular Ca2+ following some insults. In the present study, the ability of chronic exposure to (-)-nicotine to reduce cytotoxicity and attenuate increases in intracellular Ca2+ caused by exposure to N-methyl-D-aspartate were examined in organotypic cultures of rat hippocampus. Cultures were exposed to nicotine (0.1-10.0 microM) for five days prior to excitotoxic insult with N-methyl-D-aspartate. Exposure to N-methyl-D-aspartate produced concentration-dependent increases in both accumulation of 45Ca and in early and delayed cell death in the CA1, CA3 and dentate gyrus regions of cultures. The CA1 region of the hippocampus displayed the greatest sensitivity to cytotoxic effects of N-methyl-D-aspartate exposure; however, this regional difference was not associated with increased accumulation of 45Ca. Prior exposure to nicotine markedly attenuated N-methyl-D-aspartate-induced early and delayed cell death in each hippocampal region at concentrations as low as 0.1microM. However, nicotine did not alter the initial N-methyl-D-aspartate-stimulated influx of 45Ca or enhance extrusion of accumulated 45Ca measured at several time-points after insult. Five days of exposure to nicotine markedly increased immunoreactivity of the Ca2+ binding protein calbindin-D28K in each region of hippocampal cultures, effects reduced by mecamylamine co-exposure. These findings suggest that the potent protective effects of chronic nicotine exposure against neuronal overexcitation are not likely attributable to attenuations of Ca2+ accumulation, but are likely related to increased buffering of accumulated Ca2+.

Synergistic neurotoxicity of opioids and human immunodeficiency virus-1 Tat protein in striatal neurons in vitro.

Human immunodeficiency virus (HIV) infection selectively targets the striatum, a region rich in opioid receptor-expressing neural cells, resulting in gliosis and neuronal losses. Opioids can be neuroprotective or can promote neurodegeneration. To determine whether opioids modify the response of neurons to human immunodeficiency virus type 1 (HIV-1) Tat protein-induced neurotoxicity, neural cell cultures from mouse striatum were initially characterized for mu and/or kappa opioid receptor immunoreactivity. These cultures were continuously treated with morphine, the opioid antagonist naloxone, and/or HIV-1 Tat (1-72) protein, a non-neurotoxic HIV-1 Tat deletion mutant (TatDelta31-61) protein, or immunoneutralized HIV-1 Tat (1-72) protein. Neuronal and astrocyte viability was examined by ethidium monoazide exclusion, and by apoptotic changes in nuclear heterochromatin using Hoechst 33342. Morphine (10nM, 100nM or 1microM) significantly increased Tat-induced (100 or 200nM) neuronal losses by about two-fold at 24h following exposure. The synergistic effects of morphine and Tat were prevented by naloxone (3microM), indicating the involvement of opioid receptors. Furthermore, morphine was not toxic when combined with mutant Tat or immunoneutralized Tat. Neuronal losses were accompanied by chromatin condensation and pyknosis. Astrocyte viability was unaffected. These findings demonstrate that acute opioid exposure can exacerbate the neurodegenerative effect of HIV-1 Tat protein in striatal neurons, and infer a means by which opioids may hasten the progression of HIV-associated dementia.

Structure-activity analysis of dynorphin A toxicity in spinal cord neurons: intrinsic neurotoxicity of dynorphin A and its carboxyl-terminal, nonopioid metabolites.

Dynorphin A [dynorphin A (1-17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both kappa opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through kappa opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure-activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing kappa opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (> or = 1 microM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 microM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1-17) > dynorphin A (1-13) congruent with dynorphin A (2-13) congruent with dynorphin A (13-17) (least toxic) > dynorphin A (1-5) ([Leu(5)]-enkephalin) or dynorphin A (1-11). Dynorphin A (1-5) or dynorphin A (1-11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3-13), dynorphin A (6-17), and dynorphin A (13-17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 microM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13-17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3-13) and (13-17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.

Effect of nicotine on cerebellar granule neuron development.

To assess the role of nicotinic cholinergic receptors (nAChR) on neuronal maturation, nAChR expression and the direct effects of nAChR activation were examined in cerebellar external granular layer (EGL) precursors isolated in vitro. Treatment of EGL neuroblasts with nicotine elicited a concentration-dependent increase in DNA content and synthesis, implying an increase in cell numbers. Pretreatment of cultures with the nAChR antagonist dihydro-beta-erythroidine (DHBE) attenuated nicotine-induced changes in DNA abundance and synthesis. Furthermore, chronic nicotine treatment for 4-7 days promoted EGL cell survival. Epibatidine but not cytisine stimulated granule neuroblast DNA synthesis and survival. Survival effects mediated by nicotine and epibatidine were attenuated by pretreating cultures with DHBE. Immunocytochemical analysis revealed that EGL neurons possessed alpha3, but not alpha4, nAChR immunoreactivity. Quantitative autoradiography was used to determine which nAChRs are present during the period of granule cell neurogenesis in vivo. On postnatal day 5, the EGL was intensely labelled by [3H]-epibatidine but virtually devoid of [3H]-A85380 binding, suggesting that a high concentration of alpha3 AChRs is present in granule neuroblasts. The pharmacology of [3H]-epibatidine displacement from EGL neurons also suggested an interaction with the alpha3-nAChR subunits. Together these data provide novel evidence that the activation of nAChRs directly affect the development of primary cerebellar neuroblasts and further suggest that the effects are mediated through the alpha3-nAChR subtype.

Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase ('enkephalinase') gene.

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271-2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of mu receptor density in crude brain homogenates of NEP knockout mice was observed, while delta- and kappa-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident-intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.

Neurotoxicity and dysfunction of dopaminergic systems associated with AIDS dementia.

Infection with the human immunodeficiency virus (HIV) selectively targets the basal ganglia resulting in loss of dopaminergic neurons. Although frequently asymptomatic, some patients may develop signs of dopamine deficiency de novo. Accordingly, they are highly susceptible to drugs that act on dopaminergic systems. Both neuroleptics and psychostimulants may exacerbate these symptoms. Experimental evidence suggests that viral proteins such as gp120 and Tat can cause toxicity to dopaminergic neurons, and this toxicity is synergistic with compounds such as methamphetamine and cocaine that also act on the dopaminergic system. In addition, other neurotransmitters that modulate dopaminergic function, such as glutamate and opioids, may also modify the susceptibility of the dopamine system to HIV. Therefore, a thorough understanding of the mechanisms that lead to this selective neurotoxicity of dopaminergic neurons would also likely lead to the development of therapeutic modalities for patients with HIV dementia.